Enhancement of msc immunomodulatory properties by treprostinil

ABSTRACT

Provided are methods for treating or preventing vasculopathy comprising administering to a subject in need thereof, ac composition comprising a mesenchymal stem cell (MSC), or a part of a culture medium that has been in contact with the MSC and comprises one or more components of the MSC, or an exosome derived from the MSC. Pharmaceutical compositions suitable for such treatment is also provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. application Ser. No.15/790,666, filed Oct. 23, 2017, which claims priority to U.S.Provisional Patent Application No. 62/411,950 filed Oct. 24, 2016, whichis hereby incorporated by reference in its entirety.

BACKGROUND

The present application relates to mesenchymal stem cells withanti-inflammatory properties, method of making such mesenchymal stemcells, materials obtained from such mesenchymal stem cells, the use ofmesenchymal stem cells and materials obtained from mesenchymal stemcells in treatment of vasculopathy. Vasculopathy includes, but is notlimited to, pulmonary arterial hypertension (PAH), other types ofpulmonary hypertension, peripheral vascular disease (PVD), critical limbischemia (CLI), coronary artery disease, and diabetic vasculopathy.

Pulmonary hypertension is a rare, progressive, and life-threateningdisease affecting the pulmonary vasculature. Specifically, pulmonaryhypertension results in increased pressure in the pulmonary vasculature,which can lead to heart failure among other outcomes. Currently,pulmonary hypertension is classified into the following groups under theWorld Health Organisation (WHO) clinical classification system (DanaPoint 2008):

Group 1: Pulmonary arterial hypertension (PAH);Group 1′: Pulmonary veno-occlusive disease (PVOD) and/or pulmonarycapillary haemangiomatosis (PCH);Group 2: Pulmonary hypertension due to left heart diseases;Group 3: Pulmonary hypertension due to lung diseases and/or hypoxemia;Group 4: Chronic thromboembolic pulmonary hypertension (CTEPH); andGroup 5: PH with unclear multifactorial mechanisms.

Pulmonary arterial hypertension is a specific type of pulmonaryhypertension and, untreated, leads to death on average within 2.8 to 5years after being diagnosed (Keily et al. (2013) BMJ 346:f2028). Anincreasing constriction of the pulmonary circulation leads to increasedstress on the right heart, which may develop into right heart failure.By definition, the mean pulmonary arterial pressure (mPAP) in a case ofchronic pulmonary hypertension is >25 mmHg at rest or >30 mmHg duringexertion (normal value <20 mmHg). The pathophysiology of pulmonaryarterial hypertension is characterized by vasoconstriction andremodeling of the pulmonary vessels. In chronic PAH there isneomuscularization of initially unmuscularized pulmonary vessels, andthe vascular muscles of the already muscularized vessels increase incircumference. This resulting increase in pulmonary arterial pressuresresults in progressive stress on the right heart, which leads to areduced output from the right heart and eventually ends in right heartfailure (M. Humbert et al., J. Am. Coll. Cardiol. 2004, 43, 13S-24S).PAH is a rare disorder, with a prevalence of 1-2 per million. Theaverage age of the patients has been estimated to be 36 years, and only10% of the patients were over 60 years of age. Distinctly more womenthan men are affected (G. E. D'Alonzo et al., Ann. Intern. Med. 1991,115, 343-349).

Standard therapies available on the market (e.g., prostacyclinanalogues, endothelin receptor antagonists, phosphodiesteraseinhibitors) are able to improve the quality of life and the exercisetolerance of patients. These medicaments can result in serious sideeffects and/or must be delivered using complicated types ofadministration. Patients are frequently on combination therapy, eitherat the outset or after a deterioration in condition followingmonotherapy. Despite the advances in treatment, there is no cure forPAH.

Thus, a need exists to develop improved therapeutic compositions andmethods for treating vasculopathy, including PAH.

SUMMARY

In one aspect, the present disclosure provides a method of treating orpreventing vasculopathy, comprising administering to a subject in needthereof a composition comprising (i) a mesenchymal stem cell (MSC), or(ii) a part of a culture medium that has been in contact with the MSCand comprises one or more components of the MSC, or (iii) an exosomederived from the MSC, wherein the MSC has been exposed ex vivo to aprostacyclin, and wherein the exposure to the prostacyclin increases theexpression of one or more anti-inflammatory factors and/or reduces theexpression of one or more pro-inflammatory factors in the MSC, comparedto a control MSC not exposed to the prostacyclin.

In some embodiments, prior to the administration, the MSC is exposed to0.3 μg/mL to 83.3 μg/mL of prostacyclin. In other embodiments, prior tothe administration, the MSC is exposed to 0.3 μg/mL to 10 μg/mL ofprostacyclin.

In some embodiments, the prostacyclin is treprostinil, a derivative or apharmaceutically acceptable salt thereof.

In some embodiments, the MSC is exposed to the prostacyclin for at least24 hours. In other embodiments, the MSC is exposed to the prostacyclinfor at least 48 hours.

In some embodiments, the vasculopathy being treated is selected from thegroup consisting of pulmonary arterial hypertension (PAH), peripheralvascular disease (PVD), critical limb ischemia (CLI), coronary arterydisease and diabetic vasculopathy.

In some embodiments, the MSC is exposed to the prostacyclinpost-expansion.

In some embodiments, the MSC exposed to the prostacyclin has a reducedexpression level of tumor necrosis factor alpha (TNFα), compared to acontrol MSC not exposed to the prostacyclin. In some embodiments, theMSC exposed to the prostacyclin has a reduced expression level ofInterleukin-4 (IL-4), compared to a control MSC not exposed to theprostacyclin.

In other embodiments, the MSC exposed to the prostacyclin has anincreased expression level of one or more anti-inflammatory factorsselected from the group consisting of IL10, IL13, IDO, iNOS, HLA andTGFβ, compared to a control MSC not exposed to the prostacyclin.

In some embodiments, the MSC exposed to the prostacyclin has anexpression level of TNFα that is at least 50% lower than that of acontrol MSC not exposed to the prostacyclin. In other embodiments, theMSC exposed to the prostacyclin has an expression level of at least oneof IL10, IL13, IDO, iNOS, HLA and TGFβ that is at least 50% higher thanthat of a control MSC not exposed to the prostacyclin.

In some embodiments, the method of the present invention comprisesadministering to a subject in need thereof a composition comprising aMSC, wherein the MSC has been exposed ex vivo to treprostinil or apharmaceutically acceptable salt thereof at a concentration of 0.3 to 10μg/mL for at least 24 hours.

In some embodiments, the method of the present invention comprisesadministering to the subject a composition comprising a part of aculture medium that has been in contact with the MSC and comprises oneor more components of the MSC, wherein the MSC has been exposed ex vivoto treprostinil or a pharmaceutically acceptable salt thereof at aconcentration of 0.3 to 10 μg/mL for at least 24 hours, and wherein theone or more components of the MSC are selected from the group consistingof an exosome, a microvesicle, a microRNA, a messenger RNA, a non-codingRNA, a mitochondria, a growth factor, and the combinations thereof.

In some embodiments, the method of the present invention comprisesadministering to a subject in need thereof a composition comprising anexosome derived from the MSC, wherein the MSC has been exposed ex vivoto treprostinil or a pharmaceutically acceptable salt thereof at aconcentration of 0.3 to 10 μg/mL for at least 24 hours.

In some embodiments, the MSC is exposed to treprostinil or apharmaceutically acceptable salt thereof having a concentration of 0.3μg/mL to 10 μg/mL for at least 24 hours.

Also provided is a method for preparing a composition comprising amesenchymal stem cell (MSC) or a culture medium that has been in contactwith the MSC and comprises one or more components of the MSC, comprisingexposing the MSC ex vivo to a prostacyclin, and wherein the exposure tothe prostacyclin increases the expression of one or moreanti-inflammatory factors and/or reduces the expression of one or morepro-inflammatory factors in the MSC, compared to a control MSC notexposed to the prostacyclin; and isolating the MSC or the culture mediumor the one or more components of the MSC.

In some embodiments, the method comprises treating the MSC with 0.3μg/mL to 50 μg/mL of prostacyclin. In other embodiments, the methodcomprises exposing the MSC to 0.3 μg/mL to 10 μg/mL of prostacyclin.

In some embodiments, the prostacyclin is treprostinil, a derivative or asalt thereof. In some embodiments, the MSC is exposed to theprostacyclin for at least 24 hours. In other embodiments, the MSC isexposed to the prostacyclin for at least 48 hours.

In some embodiments, the MSC is exposed to the prostacyclinpost-expansion.

In some embodiments, the MSC exposed to the prostacyclin has a reducedexpression level of TNFα, compared to a control MSC not exposed to theprostacyclin. In other embodiments, the MSC exposed to the prostacyclinhas an increased expression level of one or more anti-inflammatoryfactors selected from the group consisting of IL10, IL13, IDO, iNOS, HLAand TGFβ, compared to a control MSC not exposed to the prostacyclin.

In some embodiments, the MSC exposed to the prostacyclin has anexpression level of TNFα that is at least 50% lower than that of acontrol MSC not exposed to the prostacyclin. In other embodiments, theMSC exposed to the prostacyclin has an expression level at least one ofIL10, IL13, IDO, iNOS, HLA and TGFβ that is at least 50% higher thanthat of a control MSC not exposed to the prostacyclin.

In some embodiments, the one or more components of the MSC is selectedfrom the group consisting of an exosome, a microvesicle, a microRNA, amessenger RNA, a non-coding RNA, a mitochondria, a growth factor, andthe combinations thereof.

In some embodiments, the MSC is exposed to treprostinil or a saltthereof having a concentration of 0.3 μg/mL to 10 μg/mL for at least 24hours.

In some embodiments, the method further comprises isolating an exosomefrom the culture medium.

Also provided is a composition comprising MSCs exposed to thetreprostinil obtained by the method of the present invention. In someembodiments, at least 50% of the MSCs in the composition have anexpression level of TNFα that is at least 50% lower than that of acontrol MSC not exposed to the prostacyclin. In some embodiments, atleast 50% of the MSCs in the composition have an expression level of atleast one of IL10, IL13, IDO, iNOS, HLA and TGFβ that is at least 50%higher than that of a control MSC not exposed to the prostacyclin.

Also provided is a composition comprising the isolated exosome obtainedby the method of the present invention.

In some embodiments, the composition further comprises at least onepharmaceutically acceptable carrier. In some embodiments, suchcomposition further comprises at least one additional therapeutic agentfor treating or preventing vasculopathy. In some embodiments, theadditional therapeutic agent for can be selected from the groupconsisting of NO stimulators (e.g., PDE5 inhibitors, such as tadalafil(Adcirca) and Soluble guanylate cyclase stimulators (Pro-SGC)),Endothelin receptor antagonists, and other prostacyclins.

BRIEF DESCRIPTION OF THE DRAWINGS

Provided as embodiments of this disclosure are drawings which illustrateby exemplification only, and not limitation.

FIG. 1 shows the results of immunophenotype analysis of human bonemarrow-derived MSC.

FIG. 2 provides representative images of MSC exposed in culture todifferent concentrations of treprostinil for 48 hours.

FIGS. 3A and 3B shows effects of exposure to treprostinil on MSCinflammation. FIG. 3(A) shows that under chronic hypoxia in vivo,circulating levels of TNFα increased 48% compared to control. FIG. 3(B)shows that exposure to treprostinil in vitro at dosage between 83.3μg/mL and 0.3 μg/mL decreased expression of the pro-inflammatorycytokine TNFα in MSC.

FIG. 4 illustrates that treprostinil exposure increased expressionlevels of anti-inflammatory factors IL 10 and IL 13.

FIG. 5 illustrates that treprostinil exposure increased expressionlevels of anti-inflammatory factors IDO1, iNOS, HLA, and TGFβ.

FIG. 6 shows a summary of the immunomodulatory effect of exposure totreprostinil of 9.3 μg/mL.

FIG. 7 shows a potential model for the immunomodulatory effect ofexposure to treprostinil on MSC. In particular, exposure to treprostinildecreased pro-inflammatory cytokine expression and increasedanti-inflammatory cytokine expression through intracellular (1) ornuclear signaling (2, 3, 4).

DETAILED DESCRIPTION

Exposing mesenchymal stem cells (MSC) to relatively low concentrationsof a prostacyclin, such as treprostinil, confers an anti-inflammatoryphenotype. By exposing MSCs to low concentrations of prostacyclin, thetherapeutic potential of the MSCs is enhanced based on their unique geneexpression patterns.

MSCs exposed to high concentration of treprostinil, e.g., 250 μg/mL,have enhanced expression of factors that promote angiogenesis in MSCs.However, exposing MSCs to high concentrations of treprostinil alsopromoted the expression of pro-inflammatory factors, which may not bedesirable for PAH treatment in some cases, and also negatively impactedcell viability. The present inventors unexpectedly discovered thatlow-concentration prostacyclin exposure (e.g., 10 μg/mL oftreprostinil), as described herein, decreases the expression ofpro-inflammatory factors and increases the expression ofanti-inflammatory factors in MSCs. For instance, exposing MSCs to lowconcentrations of treprostinil significantly decreased expression ofTNFα in MSCs exposed to treprostinil and significantly increased theexpression of IL-10 and IL-13. Exposing MSC to low concentrations oftreprostinil also increased expression of other anti-inflammatoryfactors, such as IDO, iNOS, HLA, and TGFβ.

MSCs exposed to a prostacyclin as described herein and products of suchMSCs can be applied as therapeutic agents. For example, MSCs may beprimed by exposing to low concentrations of prostacyclin to becomeanti-inflammatory prior to administration to a patient. As anotherexample, exosomes from MSCs treated with an appropriate concentration ofa prostacyclin, such as treprostinil, can be administered to treatvasculopathy, such as PAH. It is further contemplated that the presentinvention can be used in a bioprocess step to alter or enhanceMSC-secreted signals, such as peptides and vesicles, which may be usedas therapeutic agents.

A. Definition

Unless otherwise specified, “a” or “an” means “one or more.”

Unless specifically defined otherwise, all technical and scientificterms used herein shall be taken to have the same meaning as commonlyunderstood by one of ordinary skill in the art (e.g., in stem cellbiology, cell culture, molecular genetics, immunology,immunohistochemistry, protein chemistry, and biochemistry).

Unless otherwise indicated, the recombinant protein, cell culture, andimmunological techniques utilized in the present disclosure are standardprocedures, well known to those skilled in the art. Such techniques aredescribed and explained throughout the literature in sources such as, J.Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons(1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, ColdSpring Harbour Laboratory Press (1989), T. A. Brown (editor), EssentialMolecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press(1991), D. M. Glover and B. D. Hames (editors), DNA Cloning: A PracticalApproach, Volumes 1-4, IRL Press (1995 and 1996), and F. M. Ausubel etal. (editors), Current Protocols in Molecular Biology, Greene Pub.Associates and Wiley-Interscience (1988, including all updates untilpresent), Ed Harlow and David Lane (editors) Antibodies: A LaboratoryManual, Cold Spring Harbour Laboratory, (1988), and J. E. Coligan et al.(editors) Current Protocols in Immunology, John Wiley & Sons (includingall updates until present), and are incorporated herein by reference.

As used herein, the term “subject” (also referred to herein as a“patient”) includes warm-blooded animals, preferably mammals, includinghumans. In a preferred embodiment, the subject is a primate. In an evenmore preferred embodiment, the subject is a human.

As used herein the terms “treating”, “treat” or “treatment” includeeliminating, ameliorating, alleviating, or abating a disease orcondition or one or more symptoms thereof, whether or not the disease orcondition is considered to be “cured” or “healed” and whether or not allsymptoms are resolved. The terms also include reducing or preventingprogression of a disease or condition or one or more symptoms thereof,impeding or preventing an underlying mechanism of a disease or conditionor one or more symptoms thereof, and achieving any therapeutic and/orprophylactic benefit.

As used herein the terms “preventing”, “prevent” or “prevention” includereducing the occurrence of a disease or condition or one or moresymptoms thereof relative to an untreated control sample, or delayingthe onset of one or more symptoms of the disease or condition relativeto the untreated control sample.

As used here, the term “pro-inflammatory factor” refers to a moleculethat generally promotes inflammatory processes or is otherwisepositively associated with inflammatory processes. Pro-inflammatoryfactors include, but are not limited to, pro-inflammatory cytokines.Pro-inflammatory factors include tumor necrosis factor alpha (TNFα),Interleukin 1 (IL-1), Interleukin 6 (IL-6), Interleukin 21 (IL-21),Monocyte Chemoattractant Protein-1 (MCP1), and Monocyte ChemoattractantProtein-5 (MCP-5).

As used here, the term “anti-inflammatory factor” refers to a moleculethat generally inhibits inflammatory processes or is otherwisepositively associated with anti-inflammatory processes.Anti-inflammatory factors include, but are not limited to,anti-inflammatory cytokines. Anti-inflammatory factors includeInterleukin 10 (IL 10), Interleukin 13 (IL13), IDO, iNOS, HLA, and TGFβ.

B. Vasculopathy

Vasculopathy includes, but is not limited to, pulmonary hypertension,including pulmonary arterial hypertension (PAH), peripheral vasculardisease (PVD), critical limb ischemia (CLI), coronary artery disease,and diabetic vasculopathy.

Although many causes and conditions are found to be associated with PAH,many of them share in common several fundamental pathophysiologicalfeatures. One important feature among these processes is dysfunction ofthe endothelium, the internal cellular layer of all vessel walls, whichis normally responsible for the production and metabolism of a largearray of substances that regulate vessel tone and repair and inhibitclot formation. In the setting of PAH, endothelial dysfunction can leadto excessive production of deleterious substances and impairedproduction of protective substances. Whether this is the primary eventin the development of PAH or part of a downstream cascade remainsunknown, but in either case it is an important factor in the progressivevasoconstriction and vascular proliferation that characterize thedisease.

The term peripheral vascular disease (PVD) refers to damage, dysfunctionor obstruction within peripheral arteries and veins. Peripheral arterydisease is the most common form of PVD. Peripheral vascular disease isthe most common disease of the arteries and is a very common conditionin the United States. It occurs mostly in people older than 50 years.Peripheral vascular disease is a leading cause of disability amongpeople older than 50 years, as well as in those people with diabetes.About 10 million people in the United States have peripheral vasculardisease, which translates to about 5% of people older than 50 years. Thenumber of people with the condition is expected to grow as thepopulation ages. Men are slightly more likely than women to haveperipheral vascular disease.

Critical limb ischemia (CLI), due to advanced peripheral arterialocclusion, is characterized by reduced blood flow and oxygen delivery atrest, resulting in muscle pain at rest and non-healing skin ulcers organgrene (Rissanen et al., Eur. J. Clin. Invest. 31:651-666 (2001);Dormandy and Rutherford, J. Vasc. Surg. 31:S1-S296 (2000)). Criticallimb ischemia is estimated to develop in 500 to 1000 per millionindividuals in one year (“Second European Consensus Document on ChronicCritical Leg Ischemia”, Circulation 84 (4 Suppl.) IV 1-26 (1991)). Inpatients with critical limb ischemia, amputation, despite its associatedmorbidity, mortality and functional implications, is often recommendedas a solution against disabling symptoms (M. R. Tyrrell et al., Br. J.Surg. 80: 177-180 (1993); M. Eneroth et al., Int. Orthop. 16: 383-387(1992)). There exists no optimal medical therapy for critical limbischemia (Circulation 84 (4 Suppl.): IV 1-26 (1991)).

Coronary artery disease (atherosclerosis) is a progressive disease inhumans wherein one or more coronary arteries gradually become occludedthrough the buildup of plaque. The coronary arteries of patients havingthis disease are often treated by balloon angioplasty or the insertionof stents to prop open the partially occluded arteries. Ultimately,these patients are required to undergo coronary artery bypass surgery atgreat expense and risk.

C. Mesenchymal Stem Cells (MSCs)

Mesenchymal stem cells (MSCs) are cells found in bone marrow, blood,dental pulp cells, adipose tissue, skin, spleen, pancreas, brain,kidney, liver, heart, retina, brain, hair follicles, intestine, lung,lymph node, thymus, bone, ligament, tendon, skeletal muscle, dermis, andperiosteum. MSC are capable of differentiating into different germ linessuch as mesoderm, endoderm, and ectoderm. Thus, MSCs are capable ofdifferentiating into a large number of cell types including, but notlimited to, adipose, osseous, cartilaginous, elastic, muscular, andfibrous connective tissues. The specific lineage-commitment anddifferentiation pathway entered into by MSCs depends upon variousinfluences, including mechanical influences and/or endogenous bioactivefactors, such as growth factors, cytokines, and/or localmicroenvironmental conditions established by host tissues. MSCs are thusnon-hematopoietic progenitor cells that divide to yield daughter cellsthat are either stem cells or are precursor cells that in time willirreversibly differentiate to yield a phenotypic cell. Examples of MSCsinclude mesenchymal precursor cells (MPCs).

As used herein, the term “stem cell” refers to self-renewing cells thatare capable of giving rise to phenotypically and genotypically identicaldaughters as well as at least one other final cell type (e.g.,terminally differentiated cells). The term “stem cells” includestotipotential, pluripotential and multipotential cells, as well asprogenitor and/or precursor cells derived from the differentiationthereof.

As used herein, the term “totipotent cell” or “totipotential cell”refers to a cell that is able to form a complete embryo (e.g., ablastocyst).

As used herein, the term “pluripotent cell” or “pluripotential cell”refers to a cell that has complete differentiation versatility, i.e.,the capacity to grow into any of the mammalian body's approximately 260cell types. A pluripotent cell can be self-renewing, and can remaindormant or quiescent within a tissue.

The term “multipotential cell” or “multipotent cell” refers to a cellthat is capable of giving rise to any of several mature cell types. Asused herein, this phrase encompasses adult or embryonic stem cells andprogenitor cells, and multipotential progeny of these cells. Unlike apluripotent cell, a multipotent cell does not have the capacity to formall of the cell types.

As used herein, the term “progenitor cell” or “precursor cell” refers toa cell that is committed to differentiate into a specific type of cellor to form a specific type of tissue.

In one embodiment, cells are enriched from a sample obtained from asubject. The terms “enriched,” “enrichment,” and variations thereof areused herein to describe a population of cells in which the proportion ofone particular cell type or the proportion of a number of particularcell types is increased when compared with the untreated population.

In one embodiment, the cells used in the present disclosure are TNAP⁺,STRO-1⁺, VCAM-1⁺, THY-1⁺, STRO-2⁺, CD45⁺, CD146⁺, 3G5⁺ or anycombination thereof.

Reference to a cell “positive” (also “+”) for a given marker means thatit may be either a low (lo or dim) or a high (bright, bri) expresser ofthat marker depending on the degree to which the marker is present onthe cell surface, where the terms relate to intensity of fluorescence orother color used in the color sorting process of the cells. Thedistinction of lo (or dim or dull) and bri will be understood in thecontext of the marker used on a particular cell population being sorted.Reference to a cell as being “negative” (or “−”) for a given marker,does not mean that the marker is not expressed at all by that cell. Itmeans that the marker is expressed at a relatively very low level bythat cell, and that it generates a very low signal when detectablylabeled. In some embodiments, “negative” can refer to a marker that isnot present or present in decreased amounts in cells that have beentreated in some fashion, such as exposure to a prostacyclin. In someembodiments, “negative” refers to a marker that is present in at least50% decreased amounts in cells that have been exposed to prostacyclinwhen compared to unexposed control sample.

When used herein the term “TNAP” is intended to encompass all isoformsof tissue non-specific alkaline phosphatase. For example, the termencompasses the liver isoform (LAP), the bone isoform (BAP) and thekidney isoform (KAP). In a preferred embodiment, the TNAP is BAP. In aparticularly preferred embodiment, TNAP as used herein refers to amolecule that can bind the STRO-3 antibody produced by the hybridomacell line deposited with ATCC on 19 Dec. 2005 under the provisions ofthe Budapest Treaty under deposit accession number PTA-7282.

Stem cells useful for the methods can be derived from adult tissue, anembryo, extraembryonic tissue, or a fetus. The term “adult” is used inits broadest sense to include a postnatal subject. In a preferredembodiment, the term “adult” refers to a subject that is postpubertal.The term, “adult” as used herein can also include cord blood taken froma female.

In some aspects, the stem cells can be progeny cells (which can also bereferred to as expanded cells). Progeny cells can be produced from thein vitro culture of the stem cells described herein. Expanded cells ofthe disclosure may have a wide variety of phenotypes depending on theculture conditions (including the number and/or type of stimulatoryfactors in the culture medium), the number of passages and the like. Incertain embodiments, the progeny cells are obtained after about 2, about3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10passages from the parental population. However, the progeny cells may beobtained after any number of passages from the parental population. Andthe progeny cells can be obtained by culturing in a suitable culturemedium.

In one embodiment, the progeny cells are obtained by isolating TNAP+cells from bone marrow using magnetic beads labelled with the STRO-3antibody, and plated in α-MEM supplemented with 20% fetal calf serum, 2mM L-glutamine and 100 μm L-ascorbate-2-phosphate.

In one embodiment, such expanded cells (at least after 5 passages) canbe TNAP−, CC9+, HLA class I+, HLA class II−, CD14−, CD19−; CD3−,CD11a−c−, CD31−, CD86− and/or CD80−. However, it is possible thatexpression of different markers may vary depending on cultureconditions. Also, while cells of these phenotypes may predominate in theexpanded cell population, a minor proportion of the cells that do nothave this phenotype(s) (for example, a small percentage of the expandedcells may be CC9−) may be present. In some embodiments, these cells willbe present in an amount that is 30%, 20%, 15%, 10%, 5%, or 1% or less ofthe total number of cells present in the expanded cell population. Inone preferred embodiment, expanded cells have the capacity todifferentiate into different cell types.

In one embodiment, an expanded cell population comprises cells whereinat least 25%, more preferably at least 50%, of the cells are CC9+.

In another embodiment, an expended cell population used in the methodsof the disclosure comprises cells wherein at least 40%, more preferablyat least 45%, of the cells are STRO-1+.

In a further embodiment, the progeny cells may express markers selectedfrom the group consisting of LFA-3, THY-1, VCAM-1, PECAM-1, P-selectin,L-selectin, 3G5, CD49a/CD49b/CD29, CD49c/CD29, CD49d/CD29, CD29, CD18,CD61, integrin beta, 6-19, thrombomodulin, CD10, CD13, SCF, PDGF-R,EGF-R, IGF1-R, NGF-R, FGF-R, Leptin-R, (STRO-2=Leptin-R), RANKL,STRO-lbright, CD146, and any combination of these markers.

In one embodiment, the progeny cells are Multipotential Expanded MSCProgeny (MEMPs) as described in WO 2006/032092. Methods for preparingenriched populations of MSC from which progeny may be derived aredescribed in WO 01/04268 and WO 2004/085630. In an in vitro context MSCswill rarely be present as an absolutely pure preparation and willgenerally be present with other cells that are tissue specific committedcells (TSCCs). WO 01/04268 refers to harvesting such cells from bonemarrow at purity levels of about 0.1% to 90%. The population comprisingMSC from which progeny are derived may be directly harvested from atissue source, or alternatively it may be a population that has alreadybeen expanded ex vivo.

For example, the progeny may be obtained from a harvested, unexpanded,population of substantially purified MSC, comprising at least about 0.1,1, 5, 10, 20, 30, 40, 50, 60, 70, 80 or 95% of total cells of thepopulation in which they are present. This level may be achieved, forexample, by selecting for cells that are positive for at least onemarker selected from the group consisting of TNAP, STRO-1^(bri) 3G5+,VCAM-1, THY-1, CD146 and STRO-2.

The MSC starting population may be derived from any one or more tissuetypes set out in WO 01/04268 or WO 2004/085630, namely bone marrow,dental pulp cells, adipose tissue and skin, or perhaps more broadly fromadipose tissue, teeth, dental pulp, skin, liver, kidney, heart, retina,brain, hair follicles, intestine, lung, spleen, lymph node, thymus,pancreas, bone, ligament, bone marrow, tendon, and skeletal muscle.

MEMPS can be distinguished from freshly harvested MSCs in that they arepositive for the marker STRO-1bri and negative for the marker Alkalinephosphatase (ALP). In contrast, freshly isolated MSCs are positive forboth STRO-1^(bri) and ALP. In a preferred embodiment of the presentdisclosure, at least 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%of the administered cells have the phenotype STRO-1^(bri) ALP−. In afurther preferred embodiment the MEMPS are positive for one or more ofthe markers Ki67, CD44 and/or CD49c/CD29, VLA-3, α3β1. In yet a furtherpreferred embodiment the MEMPs do not exhibit TERT activity and/or arenegative for the marker CD18.

In one embodiment, the cells are taken from a patient with vasculopathy,cultured in vitro using standard techniques during at least a portion ofthe culturing period the cells are exposed to a prostacyclin asdescribed herein, and administered to a patient as an autologous orallogeneic transplant. In an alternative embodiment, cells of one ormore of the established human cell lines are used. In another usefulembodiment of the disclosure, cells of a non-human animal (or if thepatient is not a human, from another species) are used.

The present technology can be practiced using cells from any non-humananimal species, including but not limited to non-human primate cells,ungulate, canine, feline, lagomorph, rodent, avian, and fish cells.Primate cells with which the disclosure may be performed include but arenot limited to cells of chimpanzees, baboons, cynomolgus monkeys, andany other New or Old World monkeys. Ungulate cells with which thedisclosure may be performed include but are not limited to cells ofbovines, porcines, ovines, caprines, equines, buffalo and bison. Rodentcells with which the disclosure may be performed include but are notlimited to mouse, rat, guinea pig, hamster and gerbil cells. Examples oflagomorph species with which the disclosure may be performed includedomesticated rabbits, jack rabbits, hares, cottontails, snowshoerabbits, and pikas. Chickens (Gallus gallus) are an example of an avianspecies with which the disclosure may be performed.

Cells can be stored before use. Methods and protocols for preserving andstoring of eukaryotic cells, and in particular mammalian cells, are wellknown in the art (cf., for example, Pollard, J. W. and Walker, J. M.(1997) Basic Cell Culture Protocols, Second Edition, Humana Press,Totowa, N.J.; Freshney, R. I. (2000) Culture of Animal Cells, FourthEdition, Wiley-Liss, Hoboken, N.J.). Any method maintaining thebiological activity of the isolated stem cells such as mesenchymalstem/progenitor cells, or progeny thereof, may be utilized in connectionwith the present disclosure. In one preferred embodiment, the cells aremaintained and stored by using cryo-preservation.

Cells can be obtained using a variety of techniques. For example, anumber of cell-sorting techniques by which cells are physicallyseparated by reference to a property associated with the cell-antibodycomplex, or a label attached to the antibody can be used. This label maybe a magnetic particle or a fluorescent molecule. The antibodies may becross-linked such that they form aggregates of multiple cells, which areseparable by their density. Alternatively the antibodies may be attachedto a stationary matrix, to which the desired cells adhere.

In a preferred embodiment, an antibody (or other binding agent) thatbinds TNAP+, STRO-1+, VCAM-1+, THY-1+, STRO-2+, 3G5+, CD45+, CD146+ isused to isolate the cells. More preferably, an antibody (or otherbinding agent) that binds TNAP+ or STRO-1+ is used to isolate the cells.

Various methods of separating antibody-bound cells from unbound cellsare known. For example, the antibody bound to the cell (or ananti-isotype antibody) can be labeled and then the cells separated by amechanical cell sorter that detects the presence of the label.Fluorescence-activated cell sorters are well known in the art. In oneembodiment, anti-TNAP antibodies and/or an STRO-1 antibodies areattached to a solid support. Various solid supports are known to thoseof skill in the art, including, but not limited to, agarose beads,polystyrene beads, hollow fiber membranes, polymers, and plastic petridishes. Cells that are bound by the antibody can be removed from thecell suspension by simply physically separating the solid support fromthe cell suspension.

Super paramagnetic microparticles may be used for cell separations. Forexample, the microparticles may be coated with anti-TNAP antibodiesand/or STRO-1 antibodies. The antibody-tagged, super paramagneticmicroparticles may then be incubated with a solution containing thecells of interest. The microparticles bind to the surfaces of thedesired stem cells, and these cells can then be collected in amagnetic-field.

In another example, the cell sample is allowed to physically contact,for example, a solid phase-linked anti-TNAP monoclonal antibodies and/oranti-STRO-1 monoclonal antibodies. The solid-phase linking can comprise,for instance, adsorbing the antibodies to a plastic, nitrocellulose, orother surface. The antibodies can also be adsorbed on to the walls ofthe large pores (sufficiently large to permit flow-through of cells) ofa hollow fiber membrane. Alternatively, the antibodies can be covalentlylinked to a surface or bead, such as Pharmacia Sepharose 6 MBmacrobeads. The exact conditions and duration of incubation for thesolid phase-linked antibodies with the stem cell containing suspensionwill depend upon several factors specific to the system employed. Theselection of appropriate conditions, however, is well within the skillof the art.

The unbound cells are then eluted or washed away with physiologic bufferafter allowing sufficient time for the stem cells to be bound. Theunbound cells can be recovered and used for other purposes or discardedafter appropriate testing has been done to ensure that the desiredseparation had been achieved. The bound cells are then separated fromthe solid phase by any appropriate method, depending mainly upon thenature of the solid phase and the antibody. For example, bound cells canbe eluted from a plastic petri dish by vigorous agitation.Alternatively, bound cells can be eluted by enzymatically “nicking” ordigesting an enzyme-sensitive “spacer” sequence between the solid phaseand the antibody. Spacers bound to agarose beads are commerciallyavailable from, for example, Pharmacia.

The eluted, enriched fraction of cells may then be washed with a bufferby centrifugation and said enriched fraction may be cryopreserved in aviable state for later use according to conventional technology, cultureexpanded and/or introduced into the patient.

D. Culture Media

A “culture medium” as used herein, encompasses (a) both a culture mediumthat contains the typical components used for culturing a MSC, such asamino acids, glucose, and various salts, with or without the MSC, and(b) a composition isolated from the culture medium, including acomposition comprising components released from the MSC during theculturing. The culture medium may contain components that are solid,liquid, gaseous or a mixture of phases and materials. Culture mediumcomponents include, but are not limited to, agar, agarose, gelatin andcollagen matrices. “Culture medium” includes material that is intendedfor use in a cell culture, even if it has not yet been contacted withcells. For example, a nutrient rich liquid prepared for bacterialculture can be a culture medium.

A “culture medium that has been in contact with MSC” refers to a culturemedium that has been in contact with a MSC (e.g., for the purpose ofculturing the MSC) and thus comprises components released from the MSC.Non-limiting examples of such released components include exosomes orother microvesicles, which can comprise messenger RNA, non-coding RNA,microRNAs, mitochondria, growth factors, or other types of bioactiveagents.

E. Microvesicles and Exosomes

MSCs can release compounds and other materials into the extracellularenvironment during growth or differentiation. In some aspects, suchmaterials include extracellular vesicles. Extracellular vesiclescomprise fragments of plasma membrane derived from various cell types.Typically, extracellular vesicles have a diameter (or largest dimensionwhere the particle is not spheroid) of between about 10 nm to about 5000nm (e.g., between about 50 nm and 1500 nm, between about 75 nm and 1500nm, between about 75 nm and 1250 nm, between about 50 nm and 1250 nm,between about 30 nm and 1000 nm, between about 50 nm and 1000 nm,between about 100 nm and 1000 nm, between about 50 nm and 750 nm, etc.).Alternative names for extracellular vesicles include, but are notlimited to, microvesicles, exosomes, ectosomses, membrane particles,exosome-like particles, and apoptotic vesicles. Unless otherwisespecified, any particular type of extracellular vesicles or combinationof types of extracellular vesicles can be used according to thisdisclosure. For example, an ectosome, exosome-like particle, orcombinations thereof can be used in place of an exosome.

Exosomes are vesicles derived from the multivesicular body sortingpathway. Recent studies show that exosomes are bioactive vesicles usefulfor intercellular communication and facilitation of the immunoregulatoryprocess. MSC exosomes can contain 20S proteasomes and numerous RNAs(messenger RNA, non-coding RNA, microRNA). In some embodiments, theexosomes are between 30 nm and 200 nm in diameter or 20 nm to 50 nm indiameter. In some embodiments, the exosomes have a density in sucrose of1.10 to 1.19 g/mL, sedimented at 100,000 g. In some embodiments, theexosome's membrane can comprise sphingomyelin, ceramide, lipid rafts,and exposed phosphatidylserine.

One aspect of the present invention provides a composition comprisingexosomes isolated from MSCs that have been exposed to a prostacyclin,such as treprostinil. Such exosomes are suitable for the treatment ofvasculopathy, including pulmonary hypertension. Generally any suitablemethod for purifying and/or enriching exosomes can be used, such asmethods comprising magnetic particles, filtration, dialysis,ultracentrifugation, ExoQuick™ (Systems Biosciences, CA, USA), and/orchromatography.

In some embodiments, exosomes are isolated by centrifugation and/orultracentrifugation. The protocol is described in, for example, Thery etal. Current Protocols in Cell Biol. (2006) 3.22. In some embodiments,exosomes are isolated by a single step size exclusion chromotography.The protocol is described in, for example, Boing et al. Journal ofExtracellular Vesicles (2014) 3:23430.

In addition to exosomes, MSC also release other bioactivemolecules/vesicles. Such molecules and vesicles include, withoutlimitation, mitochondria and growth factors. Method of preparing culturemedia that contain such molecules and vesicles released from MSC andfurther isolating particular molecules and vesicles are known in theart. See Hu et al., Frontiers in Genetics, 2:56, 1-9 (2012).

F. Prostacyclin

The term “prostacyclin” used herein explicitly comprises anyprostaglandin I₂ (PGI₂), any prostacyclin analogues, and any PGI₂receptor agonists. Non-limiting examples of prostacyclins suitable forthe present technology include epoprostenol, treprostinil, iloprost, andselexipag, as well as any salts thereof, including treprostinil sodium.In one aspect, the prostacyclin is treprostinil, a derivative, apharmaceutically acceptable salt, or an ester thereof.

G. Exposing MSC to Prostacyclin

In some embodiments, prior to administration, an MSC or a culture mediumthat has been in contact with MSC can be exposed to prostacyclin.Accordingly, also provided, in some embodiments, is a method forpreparing an MSC or a culture medium that has been in contact with MSC,or an exosome derived from the MSC for in vivo delivery, comprisingcontacting the MSC or MSC-conditioned culture medium with aprostacyclin. Yet another embodiment provides a MSC or MSC-conditionedculture medium obtained by such a method.

Exposure of a cell or a medium with a chemical compound encompassesknown techniques. In one aspect, the prostacyclin can be added to andco-incubated with a culture medium that contains a MSC. Optionally,however, such co-incubation can further involve the addition of a growthfactor (e.g., VEGF and Angiopoietin-1 or -2, platelet-derived growthfactor) and/or hypoxia.

MSCs or a culture medium that has been in contact with MSCs can beexposed to prostacyclin in various ways. For example, MSCs can beexposed to prostacyclin ex vivo during the expansion of MSCs. MSCs canalso be exposed to prostacyclin post-expansion. According to oneembodiment of the present disclosure, MSCs can be prepared from thesubject's own blood or bone marrow. In that case, MSCs can be exposed toprostacyclin before they are isolated from the subject and/or the MSCscan be exposed to prostacyclin after isolation.

In some embodiments, a MSC is exposed to prostacyclin ex vivo for atleast about 12 hours, at least about 18 hours, at least about 24 hours,at least about 30 hours, at least about 36 hours, about 42 hours, atleast about 48 hours, or at least about 54 hours.

In some embodiments, a MSC is exposed to prostacyclin ex vivo atconcentration of about 0.001 μg/mL to about 100 μg/mL, about 0.001 μg/mLto about 50 μg/mL, about 0.01 μg/mL to about 20 μg/mL, about 0.1 μg/mLto about 10 μg/mL, or about 1 μg/mL to about 5 μg/mL. In someembodiments, a MSC is treated with prostacyclin at concentration ofabout 0.3 μg/mL to about 83.3 μg/mL, or about 0.3 μg/mL to about 10μg/mL. In some embodiments, a MSC is exposed to prostacyclin atconcentration of about 100 μg/mL, 75 μg/mL, 50 μg/mL, 25 μg/mL, 10μg/mL, 5 μg/mL, 2.5 μg/mL, 1 μg/mL, 0.5 μg/mL, 0.25 μg/mL, 0.1 μg/mL,0.05 μg/mL, or about 0.01 μg/mL. The concentration of prostacyclinrefers to the concentration of prostacyclin in the MSC culture medium.

In some embodiments, MSC exposed to prostacyclin has an expression levelof a pro-inflammatory factor or pro-inflammatory cytokine that is atleast 30%, at least 40%, at least 50%, at least 1 fold, at least 1.5fold, at least 2 fold, at least 3 fold, at least 4 fold, or at least 5fold lower than the expression level of a control MSC not exposed to theprostacyclin. In some embodiments, the pro-inflammatory factor is TNF-α.In some embodiments, the expression level of more than onepro-inflammatory factors are decreased by the prostacyclin exposure.

In some embodiments, MSC exposed to prostacyclin has an expression levelof an anti-inflammatory factor or anti-inflammatory cytokine that is atleast 30%, at least 40%, at least 50%, at least 1 fold, at least 1.5fold, at least 2 fold, at least 3 fold, at least 4 fold, or at least 5fold, at least 6 fold, at least 7 fold, at least 8 fold, at least 9fold, or at least 10 fold higher than the expression level of a controlMSC not exposed to the prostacyclin. In some embodiments, the expressionlevel of more than one anti-inflammatory factors are increased by theprostacyclin exposure. In some embodiments, the anti-inflammatory factoris selected from the group consisting of IL 10, IL13, IDO, iNOS, HLA,TGFβ, and a combination thereof.

In some embodiments, exposure of MSC to prostacyclin simultaneouslyreduces the expression level of one or more pro-inflammatory factors,and increases the expression level of one or more anti-inflammatoryfactors. In some embodiments, exposure of MSC to prostacyclinsimultaneously reduces the expression level of TNF-α and/or IL-4, andincreases the expression level of one or more anti-inflammatory factorsselected from the group consisting of IL 10, IL13, DO, iNOS, HLA, andTGFβ.

H. Pharmaceutical Compositions

In one aspect, the present disclosure provides a pharmaceuticalcomposition comprising a therapeutically effective amount of a MSC, or apart of a culture medium that has been in contact with the MSCcomprising one or more components of the MSC, or exosomes derived fromthe MSC.

In some embodiments, the pharmaceutical composition comprises MSCs thathave been exposed to treprostinil, such as those described herein. Insome embodiments, at least 30%, at least 40%, at least 50%, at least60%, at least 70%, at least 80%, at least 90%, at least 95%, at least98%, or at least 99% of all MSCs in the composition have an expressionlevel of TNFα that is at least 50% lower than that of a control MSC notexposed to the prostacyclin. In some embodiments, at least 30%, at least40%, at least 50%, at least 60%, at least 70%, at least 80%, at least90%, at least 95%, at least 98%, or at least 99% of all MSCs in thecomposition have an expression level of at least one of IL10, IL13, IDO,iNOS, HLA and TGFβ that is at least 50% higher than that of a controlMSC not exposed to the prostacyclin.

In some embodiments, the pharmaceutical composition further comprises atleast one pharmaceutically-acceptable carrier. The phrase“pharmaceutically acceptable” refers to those compounds, materials,compositions, and/or dosage forms which are, within the scope of soundmedical judgment, suitable for use in contact with the tissues of humanbeings and animals without excessive toxicity, irritation, allergicresponse, or other problem or complication, commensurate with areasonable benefit/risk ratio. The phrase “pharmaceutically-acceptablecarrier” as used herein means a pharmaceutically-acceptable material,composition or vehicle, such as a liquid or solid filler, diluent,excipient, or solvent encapsulating material.

Pharmaceutically acceptable carriers include saline, aqueous buffersolutions, solvents and/or dispersion media. The use of such carriersare well known in the art. The solution is preferably sterile and fluidto the extent that easy syringability exists. Preferably, the solutionis stable under the conditions of manufacture and storage and preservedagainst the contaminating action of microorganisms such as bacteria andfungi through the use of, for example, parabens, chlorobutanol, phenol,ascorbic acid, thimerosal, and the like.

Some examples of materials and solutions which can serve aspharmaceutically-acceptable carriers include: (1) sugars, such aslactose, glucose and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4)powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients,such as cocoa butter and suppository waxes; (9) oils, such as peanutoil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters,such as ethyl oleate and ethyl laurate; (13) agar; (14) bufferingagents, such as magnesium hydroxide and aluminum hydroxide; (15) alginicacid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer'ssolution; (19) ethyl alcohol; (20) pH buffered solutions; (21)polyesters, polycarbonates and/or polyanhydrides; and (22) othernon-toxic compatible substances employed in pharmaceutical formulations.

The pharmaceutical compositions useful for the methods of the disclosuremay comprise a polymeric carrier or extracellular matrix.

A variety of biological or synthetic solid matrix materials (e.g., solidsupport matrices, biological adhesives or dressings, andbiological/medical scaffolds) are suitable for use in this disclosure.The matrix material is preferably medically acceptable for use in invivo applications. Non-limiting examples of such medically acceptableand/or biologically or physiologically acceptable or compatiblematerials include, but are not limited to, solid matrix materials thatare absorbable and/or non-absorbable, such as small intestine submucosa(SIS), e.g., porcine-derived (and other SIS sources); crosslinked ornon-crosslinked alginate, hydrocolloid, foams, collagen gel, collagensponge, polyglycolic acid (PGA) mesh, polyglactin (PGL) mesh, fleeces,foam dressing, bioadhesives (e.g., fibrin glue and fibrin gel) and deadde-epidermized skin equivalents in one or more layers

Suitable polymeric carriers include porous meshes or sponges formed ofsynthetic or natural polymers, as well as polymer solutions. One form ofmatrix is a polymeric mesh or sponge; the other is a polymeric hydrogel.Natural polymers that can be used include proteins such as collagen,albumin, and fibrin; and polysaccharides such as alginate and polymersof hyaluronic acid. Synthetic polymers include both biodegradable andnon-biodegradable polymers. Examples of biodegradable polymers includepolymers of hydroxy acids such as polylactic acid (PLA), polyglycolicacid (PGA), and polylactic acid-glycolic acid (PLGA), polyorthoesters,polyanhydrides, polyphosphazenes, and combinations thereof.Non-biodegradable polymers include polyacrylates, polymethacrylates,ethylene vinyl acetate, and polyvinyl alcohols.

Polymers that can form ionic or covalently crosslinked hydrogels whichare malleable are used to encapsulate cells. A hydrogel is a substanceformed when an organic polymer (natural or synthetic) is cross-linkedvia covalent, ionic, or hydrogen bonds to create a three-dimensionalopen-lattice structure which entraps water molecules to form a gel.Examples of materials which can be used to form a hydrogel includepolysaccharides such as alginate, polyphosphazines, and polyacrylates,which are crosslinked ionically, or block copolymers such as Pluronics™or Tetronics™, polyethylene oxide-polypropylene glycol block copolymerswhich are crosslinked by temperature or pH, respectively. Othermaterials include proteins such as fibrin, polymers such aspolyvinylpyrrolidone, hyaluronic acid and collagen.

In general, these polymers are at least partially soluble in aqueoussolutions, such as water, buffered salt solutions, or aqueous alcoholsolutions, that have charged side groups, or a monovalent ionic saltthereof. Examples of polymers with acidic side groups that can bereacted with cations are poly(phosphazenes), poly(acrylic acids),poly(methacrylic acids), copolymers of acrylic acid and methacrylicacid, poly(vinyl acetate), and sulfonated polymers, such as sulfonatedpolystyrene. Copolymers having acidic side groups formed by reaction ofacrylic or methacrylic acid and vinyl ether monomers or polymers canalso be used. Examples of acidic groups are carboxylic acid groups,sulfonic acid groups, halogenated (preferably fluorinated) alcoholgroups, phenolic OH groups, and acidic OH groups. Examples of polymerswith basic side groups that can be reacted with anions are poly(vinylamines), poly(vinyl pyridine), poly(vinyl imidazole), and some iminosubstituted polyphosphazenes. The ammonium or quaternary salt of thepolymers can also be formed from the backbone nitrogens or pendant iminogroups. Examples of basic side groups are amino and imino groups.

In some embodiments, the pharmaceutical composition can comprise atleast one additional therapeutic agent. For example, the composition maycontain an analgesic to aid in treating inflammation or pain, or ananti-infective agent to prevent infection of the site treated with thecomposition. More specifically, non-limiting examples of usefultherapeutic agents include the following therapeutic categories:analgesics, such as nonsteroidal anti-inflammatory drugs, opiateagonists and salicylates; anti-infective agents, such as antihelmintics,antianaerobics, antibiotics, aminoglycoside antibiotics, antifungalantibiotics, cephalosporin antibiotics, macrolide antibiotics,miscellaneous β-lactam antibiotics, penicillin antibiotics, quinoloneantibiotics, sulfonamide antibiotics, tetracycline antibiotics,antimycobacterials, antituberculosis antimycobacterials, antiprotozoals,antimalarial antiprotozoals, antiviral agents, anti-retroviral agents,scabicides, anti-inflammatory agents, corticosteroid anti-inflammatoryagents, antipruritics/local anesthetics, topical anti-infectives,antifungal topical anti-infectives, antiviral topical anti-infectives;electrolytic and renal agents, such as acidifying agents, alkalinizingagents, diuretics, carbonic anhydrase inhibitor diuretics, loopdiuretics, osmotic diuretics, potassium-sparing diuretics, thiazidediuretics, electrolyte replacements, and uricosuric agents; enzymes,such as pancreatic enzymes and thrombolytic enzymes; gastrointestinalagents, such as antidiarrheals, gastrointestinal anti-inflammatoryagents, gastrointestinal anti-inflammatory agents, antacid anti-ulceragents, gastric acid-pump inhibitor anti-ulcer agents, gastric mucosalanti-ulcer agents, H2-blocker anti-ulcer agents, cholelitholyticagent's, digestants, emetics, laxatives and stool softeners, andprokinetic agents; general anesthetics, such as inhalation anesthetics,halogenated inhalation anesthetics, intravenous anesthetics, barbiturateintravenous anesthetics, benzodiazepine intravenous anesthetics, andopiate agonist intravenous anesthetics; hormones and hormone modifiers,such as abortifacients, adrenal agents, corticosteroid adrenal agents,androgens, anti-androgens, immunobiologic agents, such asimmunoglobulins, immunosuppressives, toxoids, and vaccines; localanesthetics, such as amide local anesthetics and ester localanesthetics; musculoskeletal agents, such as anti-gout anti-inflammatoryagents, corticosteroid anti-inflammatory agents, gold compoundanti-inflammatory agents, immunosuppressive anti-inflammatory agents,nonsteroidal anti-inflammatory drugs (NSAIDs), salicylateanti-inflammatory agents, minerals; and vitamins, such as vitamin A,vitamin B, vitamin C, vitamin D, vitamin E, and vitamin K.

Compositions useful for the methods of the present disclosure mayinclude cell culture components, e.g., culture media including aminoacids, metals, coenzyme factors, as well as small populations of othercells, e.g., some of which may arise by subsequent differentiation ofthe stem cells.

Compositions useful for the methods of the present disclosure may beprepared, for example, by sedimenting out the subject cells from theculture medium and re-suspending them in the desired solution ormaterial. The cells may be sedimented and/or changed out of the culturemedium, for example, by centrifugation, filtration, ultrafiltration,etc.

I. Administration

In some embodiments, the pharmaceutical composition can be administeredalone or co-administered with prostacyclin. In some embodiments, thepharmaceutical composition and prostacyclin are administeredconcurrently. In other embodiments, the prostacyclin and the compositionare administered separately. When administered separately, theprostacyclin can be administered prior to, or following theadministration of the MSC composition.

The skilled artisan can readily determine the amount of cells andoptional carrier(s) in compositions and to be administered in methods ofthe disclosure. In an embodiment, any additives (in addition to theactive cell(s)) are present in an amount of 0.001 to 50% (weight)solution in phosphate buffered saline, and the active ingredient ispresent in the order of micrograms to milligrams, such as about 0.0001to about 5 wt %, preferably about 0.0001 to about 1 wt %, still morepreferably about 0.0001 to about 0.05 wt % or about 0.001 to about 20 wt%, preferably about 0.01 to about 10 wt %, and still more preferablyabout 0.05 to about 5 wt %. Of course, for any composition to beadministered to an animal or human, and for any particular method ofadministration, it is preferred to determine therefore: toxicity, suchas by determining the lethal dose (LD) and LD50 in a suitable animalmodel e.g., rodent such as mouse; and, the dosage of the composition(s),concentration of components therein and timing of administering thecomposition(s), which elicit a suitable response. Such determinations donot require undue experimentation from the knowledge of the skilledartisan, this disclosure and the documents cited herein. And, the timefor sequential administrations can be ascertained without undueexperimentation.

Compositions useful for the methods of the present disclosure can beadministered via, inter alia, localized injection, including catheteradministration, systemic injection, localized injection, intravenousinjection, intrauterine injection or parenteral administration. Whenadministering a therapeutic composition described herein (e.g., apharmaceutical composition), it will generally be formulated in a unitdosage injectable form (solution, suspension, emulsion).

According to one embodiment of the present disclosure, the compositionscan be co-administered with at least one other medicine forvasculopathy. For example, the pharmaceutical compositions can beco-administered with a prostaglandin I₂ (PGI₂), prostacyclin analogues,phosphodiesterase-5 (PDE-5) inhibitor, endothelin receptor antagonist(ETRA), tyrosine kinase inhibitors, or soluble guanylate cyclasestimulator.

According to one embodiment, the method for treating vasculopathyfurther comprises reducing thrombosis in pulmonary arteries, reducinginflammation in pulmonary arteries, reducing the proliferation ofintimal smooth muscle in pulmonary arteries, reducing the formation ofplexiform lesions in pulmonary arteries, increasing the amount of nitricoxide in pulmonary arteries, increasing the amount of PGI2 in pulmonaryarteries, reducing the level of Endothelin-1 in pulmonary arteries,reducing the amount of growth factors in pulmonary arteries, orpromoting proper endothelial morphology in pulmonary arteries.

Although the foregoing refers to particular preferred embodiments, itwill be understood that the present disclosure is not so limited. Itwill occur to those of ordinary skill in the art that variousmodifications may be made to the disclosed embodiments and that suchmodifications are intended to be within the scope of the presentdisclosure.

All of the publications, patent applications and patents cited in thisspecification are incorporated herein by reference in their entirety. Tothe extent that these publications, patent applications and patentscontain definitions that differ from the definition provided herein oruses terms or phrases in a different manner, the definitions and usagesin this specification control.

Examples

The following examples are intended to provide those of ordinary skillin the art with a complete disclosure and description of how to make anduse the methods and compositions described herein, and are not intendedto be limiting.

Example 1—MSC Phenotype and Morphology Studies

This example verifies the phenotype of the MSC cells exposed totreprostinil and determines an optimal dose range for treprostinilexposure without producing cytotoxic effects on the MSC cell.

A single vial of human bone marrow-derived MSC was expanded and seededusing standard growth medium. At 95-99% confluency, cells werethoroughly washed with phosphate-buffered saline (PBS) and exposed tomedia containing treprostinil at doses ranging from 250 μg/mL to 0.004μg/mL. After 48 hours of culture, cells were photographed and assessedfor treatment-induced changes in morphology.

Flow cytometry analysis (FIG. 1 ) demonstrated that the bone marrow MSCsused in this study were negative or low for CD34, CD45, and HLA-DR andpositive for MSC markers CD73, CD105, and CD90. Definition of MSC wasestablished by the International Society for Cellular Therapy (Dominiciet al., Cytotherapy 8(4):315-7, 2006).

Images of MSCs exposed to different concentrations of treprostinil (FIG.2 ) showed that MSCs appear rounded up and detached at the highest Tredose (250 μg/mL), which is indicative of a cytotoxic effect oftreprostinil on MSC. On the other hand, treprostinil at doses 83.3 μg/mLor lower did not cause morphological changes associated with cell death.

This example demonstrates that high doses of treprostinil negativelyimpact MSC cell viability while doses of treprostinil lower than 83.3μg/mL did not cause this cytotoxic effect (FIG. 2 ). Subsequent studiesincluded a range of low treprostinil doses to avoid cytotoxic effects.

Example 2— Treprostinil Effects on Pro- and Anti-Inflammatory Cytokines

This example examined the effect of treprostinil on the production ofpro- and anti-inflammatory cytokines in MSC.

Cells exposed to treprostinil ranging from 250 μg/mL-0.004 μg/mL asdescribed in Example 1 were harvested. RNA of the cells was extractedand then analyzed for pro- and anti-inflammatory cytokines. Cytokines,or small proteins involved in both internal and secreted cell signaling,have been identified to play a role in the inflammatory pathogenesis ofPAH (Groth et al., Respiratory Research, 15:47 (2014)).

Under chronic hypoxia in vivo, circulating levels of TNFα increased 48%compared to control (FIGS. 3A and 3B). In vitro treprostinil exposure atdoses between 83.3 and 0.3 μg/ml decreased the expression level of thepro-inflammatory cytokine TNFα in MSC.

Exposure of MSC to increasing doses of treprostinil revealed an increasein IL10 and IL13 signaling over control (FIG. 4 ).

In hypoxia-induced PAH mice, a 48% increase in circulating levels ofTNFα was observed (FIG. 3A). Interestingly, exposure with treprostinilof 0.3 μg/mL to 83.3 μg/mL lowered the expression of TNFα by up to 4.7fold compared to the control (FIG. 3B). Further analysis revealed thatthe same treprostinil dose range increased the expression level ofanti-inflammatory cytokines IL10 and IL13. Specifically, the expressionof IL10 in MSC increased by 5.4 fold when exposed to 9.3 μg/mLtreprostinil. The expression of IL13 in MSC increased by 8.8 fold whenexposed to 27.8 μg/mL treprostinil (FIG. 4 ). These data indicates thatthe anti-inflammatory potential of MSCs was induced by low-concentrationtreprostinil exposure.

Example 3— Treprostinil Effects on Production of ImmunosuppressiveFactors

This example examined the effect of treprostinil on the MSC productionof several immunosuppressive factors.

RNA from MSCs exposed to treprostinil of from 250 μg/mL to 0.004 μg/mLwas re-analyzed for several other anti-inflammatory factors. Genes thathave been shown to play a specific role in the immunosuppressiveproperties of MSC including IDO1, iNOS, HLA and TGFβ (Hoogduijn et al.,International Immunopharmacology, 1496-1500(2010)) were assessed.

MSC exposed to increasing doses of treprostinil revealed a robustincrease in anti-inflammatory factors IDO1, iNOS, HLA and TGFβ overcontrol (FIG. 5 ).

Exposure of MSC to 9.3 μg/mL treprostinil decreased TNFα(pro-inflammatory cytokine) and increased IL10, IL13, IDO, iNOS, HLA andTGFβ (anti-inflammatory factors) over unexposed control (FIG. 6 ).

This example demonstrates that MSC exposed to increasing doses oftreprostinil revealed a robust increase in anti-inflammatory factorsIDO1, iNOS, HLA and TGFβ over control (FIG. 5 ). This gene expressionprofile, coupled with the cytokine profile in Example 2 indicate analteration in MSC gene expression pattern after 48 hours of exposure tomultiple doses of treprostinil in vitro. Exposure to 9.3 μg/mLtreprostinil decreased the pro-inflammatory cytokine TNFα and increasedseveral anti-inflammatory factors including IL10, IL13, IDO, iNOS, HLAand TGFβ (FIG. 6 ). These data suggest treprostinil-exposed MSC couldserve as an immunomodulatory factor in the treatment of disease.

A proposed model for the immunomodulatory effect of treprostinilexposure is illustrated in FIG. 7 . In particular, treprostinil exposureof MSC decreased pro-inflammatory and increased anti-inflammatorycytokines through intracellular (1) or nuclear signaling (2, 3, 4).Further analysis revealed genetic reprogramming of treprostinil-exposedMSC toward an anti-inflammatory state through nuclear signaling (2)measured by changes in RNA expression (3).

1. A method for preparing a composition comprising a mesenchymal stemcell (MSC) or a culture medium that has been in contact with the MSC andcomprises one or more components of the MSC, comprising exposing the MSCto a prostacyclin ex vivo, and wherein the exposure with theprostacyclin increases the expression of one or more anti-inflammatoryfactors and/or reduces the expression of one or more pro-inflammatoryfactors in the MSC, compared to a control MSC not exposed to theprostacyclin; and isolating the MSC or at least a portion of the culturemedium comprising exosomes.
 2. The method of claim 1, comprisingexposing the MSC to 0.3 μg/mL to 50 μg/mL of prostacyclin.
 3. The methodof claim 1, comprising exposing the MSC to 0.3 μg/mL to 10 μg/mL ofprostacyclin.
 4. The method of claim 1, wherein the prostacyclin istreprostinil, a derivative or a salt thereof.
 5. The method of claim 1,wherein the MSC is exposed to the prostacyclin for at least 24 hours. 6.The method of claim 1, wherein the MSC is exposed to the prostacyclinfor at least 48 hours.
 7. The method of claim 1, wherein the MSC isexposed to the prostacyclin post-expansion.
 8. The method of claim 1,wherein the MSC exposed to the prostacyclin has a reduced expressionlevel of TNFα, compared to a control MSC not exposed to theprostacyclin.
 9. The method of claim 1, wherein the MSC exposed to theprostacyclin has an increased expression level of one or moreanti-inflammatory factors selected from the group consisting of IL10,IL13, IDO, iNOS, HLA and TGFβ, compared to a control MSC not exposed tothe prostacyclin.
 10. The method of claim 1, wherein the MSC exposed tothe prostacyclin has an expression level of TNFα that is at least 50%lower than that of a control MSC not exposed to the prostacyclin. 11.The method of claim 1, wherein the MSC exposed to the prostacyclin hasan expression level at least one of IL10, IL13, IDO, iNOS, HLA and TGFβthat is at least 50% higher than that of a control MSC not exposed tothe prostacyclin.
 12. The method of claim 1, wherein the one or morecomponents of the MSC is selected from the group consisting of anexosome, a microvesicle, a microRNA, a messenger RNA, a non-coding RNA,a mitochondria, a growth factor, and the combinations thereof.
 13. Themethod of claim 1, wherein the MSC is exposed to a compositioncomprising treprostinil or a salt thereof having a concentration of 0.3μg/mL to 10 μg/mL for at least 24 hours.
 14. The method of claim 1,further comprising isolating an exosome from the culture medium.
 15. Acomposition comprising the isolated MSC obtained by the method ofclaim
 1. 16. A composition comprising the isolated exosome obtained bythe method of claim
 1. 17. The composition of claim 15, furthercomprising at least one pharmaceutically acceptable carrier.
 18. Thecomposition of claim 15, further comprising at least one additionaltherapeutic agent for treating or preventing vasculopathy.
 19. Thecomposition of claim 16, further comprising at least onepharmaceutically acceptable carrier.
 20. The composition of claim 16,further comprising at least one additional therapeutic agent fortreating or preventing vasculopathy.
 21. A composition comprising apopulation of MSCs having an altered gene expression pattern, whereinthe altered gene expression pattern comprises an expression level ofTNFα that is at least 50% lower than that of a control MSC and/or anexpression level of at least one of IL10, IL13, IDO, iNOS, HLA and TGFβthat is at least 50% higher than that of a control MSC, wherein thepopulation of MSCs having said altered gene expression pattern accountsfor at least 50% of all MSCs in the composition.